Monoclonal antibody producing hybridoma and corresponding antibody specific for human breast fibroblast tumor antigen

ABSTRACT

The invention concerns a novel lymphocyte hybridoma and an antibody which may be generated from the hybridoma. The hybridoma and antibody have the internal designation LICR-LON-Fib 75. The origin, method of preparation and uses are discussed. The antibody has particular application in the diagnosis and treatment of cancer of the breast. A particular therapeutic treatment comprises harvesting a sample of bone marrow from a patient, subjecting the patient to treatment adapted to kill cancerous material including that within the bone marrow (possibly including the normal differentiated haemopoietic cells of the marrow), subjecting some or all of the sample to cytotoxic treatment with the antibody (or a toxin conjugate thereof) adapted to kill cancerous cell lines while leaving viable colony-forming units of bone marrow, and reintroducing the treated sample material to the blood stream of the patient.

This is a continuation of copending application Ser. No. 558,538, filedon Dec. 5, 1983 now U.S. Pat. No. 4,731,238 issued Mar. 15, 1988.

The present invention relates to a novel monoclonal hybridoma, which maybe stored as a tissue culture, and to the corresponding antibody whichmay be generated from the hybridoma and which has important applicationsin the diagnosis and treatment of cancer, particularly cancer of thebreast.

The preferred hybridoma has the internal designation LICR-LON-Fib 75. Itis available at the Collection Nationale de Cultures deMicro-Organismes, Institut Pasteur, 128, Rue de Docteur Roux 75724Paris, France, under the designation CNCM, I-211 (deposit date Dec. 8,1982).

This invention also includes the antibody expressed by such hybridoma,the antigen specific to the antibody and equivalent structures as setout in the claims forming a part of this specification.

LYMPHOCYTE HYBRIDOMAS

The preferred hybridoma and antibody of this invention, are hereafterreferred to shortly as Fib-75 and will have the subscripts H forhybridoma and A for antibody. Fib-75-H is a lymphocyte hybridomaobtained by fusing myeloma cells with normal antibody-secreting cells.Such hybrids are fully discussed in Ref. 1. As discussed in theintroduction to that paper, the myelomas are a group of human tumourswhich have long been considered to be derived by the proliferation ofsingle clones of antibody-forming cells. Naturally-occurring myelomatumours frequently produce an intact antibody, or specific fragmentsthereof, although, occasionally, myelomas which are unable to secreteimmunoglobulins are also encountered. The demonstration that ananalogous tumour could be induced in mice provided the opportunity toproduce unlimited amounts of homogeneous immunoglobulin together with amodel system with which to investigate the synthesis of antibodies.However, the generation of myelomas making a specific antibody against apre-defined antigen has never been achieved by this technique. Thesolution to this problem became apparent in 1975 when Kohler andMilstein (Ref.2) demonstrated that, by fusing myeloma cells with normalantibody-secreting cells, they generated a range of lymphocytehybridomas which produced antibodies and which, being tumours,immortalized the antigen specificity of the normal parent cells.

The power of the lymphocyte hybridoma system, as devised by Kohler andMilstein, lies in the ability to raise, select and continuously generatespecific antibodies to individual determinants following immunizationwith a heterogeneous and undefined immunogen. The system is extremelysensitive and avoids the initial isolation of a possibly usefulantigen--an essential procedure in the production of conventionalantisera. It also avoids the numerous steps of adsorption which arefrequently required in the production of a conventional heteroantiserumof the desired specificity.

The primary objective of fusing somatic cells with an appropriate cellline is the generation of karyotypically stable hybrids whichimmortalize specific functions (phenotypes) expressed by normaldifferential cells. The characteristic phenotype of B-lymphocyteshappens to be the generation of identifiable and usefulproducts--antibodies. Similarly, T-lymphocytes express characteristicsurface determinants, and somatic cells of non-lymphoid origin arerecognized by particular isoenzymes or other specific features. Detailsof the technical aspects of producing somatic-cell hybrids andgenerating monoclonal antibodies have recently been reviewed by Galfreand Milstein (Ref.3).

In this specification, the term "determinant" is used synonymously withthat of "epitope" to define the precise region or structuralconfiguration of a molecule identified by a monoclonal antibody. Theterm "antigen" will be reserved to define a complete molecule identifiedby a conventional heteroantiserum and which may comprise multipleepitopes, each able to be identified by a different monoclonal antibody.

The steps taken in the preparation of the hybridoma and the generationof the monoclonal antibody are conveniently summarized in FIG. 1 of Ref.1 as shown in FIG. 1 of U.S. Pat. No. 4,731,238.

ORIGIN AND GENERATION OF FIB 75

Fib 75 was isolated as part of a study undertaken with the aim ofraising a panel of mouse monoclonal antibodies to human breastfibroblasts. The objectives were to select antibodies which couldidentify human breast fibroblasts in culture and which (if possible)could be used as cytotoxic reagents. For these purposes, BALB/c micewere immunised with cells obtained from primary monolayer cultures ofnormal human breast tissue.

A number of hybridomas were isolated in the course of this study inrespect of which details have been published in the 1981 Annual Reportof the Ludwig Institute For Cancer Research (London Branch) and in thepublished papers identified hereafter as Refs. 4, 5, 6. As describedparticularly in Ref. 4 the mouse myeloma utilized was that designatedNS1, originating with Dr. C. Milstein (Cambridge, UK). A number of thehybridomas and corresponding antibodies isolated in the course of thestudy are now well known. However, Fib 75, although referred to e.g. inthe aforesaid Annual Report, has not been publicly available before thedate of this application.

The general steps of preparation are now described and these followgenerally the scheme shown in the drawing.

(i) Preparation of Human Breast Epithelial Organoids

Normal human breast tissues were obtained from a first female patientfollowing reduction mammoplasty surgery performed for cosmetic reasons.So-called `normal` breast tissues from those regions of mastectomyspecimens uninvolved by malignancy were specifically excluded. Attachedskin and excess fat were trimmed from the specimens using scissors andthe residual tissues diced into pieces no larger than 5 mm cubes. Themacerated breast tissues were digested with four volumes of Dulbecco'sminimal essential medium (DMEM) containing 0.4 mg/milliliter collagenase(Type 1A, Sigma, Poole, U.K.). Kanamycin (100 μg/ml) and gentamycin (80μg/ml) were included at all stages of the preparative procedures. Breastepithelial organoids liberated from the intact normal tissues werecollected by centrifugation. Precise details of the method have beendescribed in Ref. 7.

(ii) Culture of breast Epithelial Organoids

The breast epithelial organoids from (i) were suspended in Dulbecco'sminimal essential medium DMEM containing 10% (v/v) foetal calf serum(Gibco) and Kanamycin (100 μg/ml). The suspensions were plated-out intocapped tissue-culture flasks (Nunc., Inter Med., 75 cm² surface area)and incubated at 37° C. in an atmosphere of 100% humidity and 5% CO₂ inair.

The DMEM medium surrounding the serum cells and organoids which hadattached during the first 48 hours was replaced on alternate days byfresh identical medium. These cultures were maintained for at least fiveweeks. At the end of this period, the cultures comprised interlacingbundles of fusiform cells (fibroblasts). Cells having a cuboidalappearance (epithelial cells) had almost totally disappeared from thesecultures.

(iii) Immunisation of Mice

Culture medium was decanted from the monolayer cell-cultures and thecells were rinsed briefly with sterile normal saline to remove adsorbedfoetal calf serum. The attached cells were scraped from theculture-flask with a clean soft rubber spatula. The cells from each 75cm² flask were suspended in 1 ml sterile normal saline. To thissuspension was added 0.5 ml Freund's complete adjuvant and an emulsionprepared using a Vortex (Trade Mark) Mixer. 0.5 ml of this mixture wasinjected, subcutaneously into each of three female BALB/c mice.

7 days after the primary immunisation, the mice were boosted by anintraperitoneal injection of such cells but obtained from a secondfemale patient and prepared in a manner similar to that alreadydescribed.

(iv) Fusion

3 days after the mice had been boosted with immunogen, the spleen fromone animal was removed, disaggregated and then fused in the presence ofpoly(ethylene glycol) (PEG) with cells of the murine myeloma NSI (Refs.3 and 4) using the method description in ref. 5. Hybrid cells wereselected using medium containing hypoxanthine, aminopterin andthyimidine according to the techniques of Littlefield (1964) (Ref.8).Hybridoma colonies were grown from the outset in semisolid 0.25% (w/v)agar.

(v) Primary Screening of Hybrids

10 days after the fusion, tissue-culture supernatants were assayed forspecific binding to cultivated breast fibroblasts. The fibroblasts(fusiform cells) were obtained from normal human breast by the methodpreviously described (see (ii) above). Cells from a third patient wereemployed as the source of these cells. One week prior to screening,cells from each 75 cm² plate were resuspended by trypsin and dividedequally between the wells of two 24-well (Costar) tissue-culture plates.The cells were cultured in DMEM and under the conditions previouslydescribed in (i) above.

Duplicate 100 μl aliquots of each hybridoma culture supernatants wereincubated with the `target` fibroblasts. Bound antibodies wereidentified using an ¹²⁵ I-labelled affinity purified rabbit anti-mouseimmunoglobulin G (Sera Lab.) (Ref.9).

Colonies from positive wells were picked from the agar and culturedseparately. 7 days later, positive colonies were identified using anidentical binding-assay.

(vi) Immunohistochemistry

Culture supernatants from positive colonies were concentrated tenfoldusing Amicon (Trade Mark) B125 filtration chambers. The concentratedsupernatants were screened for immunohistochemical binding to normalhuman tissues using the indirect immunophosphatase technique (Ref.5,p.281(v), Ref.7).

(vii) Selection and Cloning of `Fib 75`

The binding assays and immunohistochemical studies indicated that theantibody designated LICR-LON-FIB75 bound to many normal human tissues,including the milk fat globule membrane of human breast origin. Singlehybridoma cells producing this antibody were cloned by limiting dilution(Ref.10) grown up as separate individual colonies in tissue-culture, andthe colonies producing the Fib-75 antibody were identified by binding tothe MFGM (human milk fat globule membrane) using the techniquepreviously described (Ref.5 p.281(iv)).

(viii) Antibody Collection and Purification

Cells of the hybridoma Fib-75-H were injected into the peritoneal cavityof BALB/c-mice previously treated with the mineral oil Pristane (TradeMark) (2,6,10,14-tetramethylpentadecane) (Ref.1. p.67). Theproliferating hybridoma cells caused the production of ascites withinthe peritoneal cavities and this was harvested as it developed.

The monoclonal antibody Fib-75-A was precipitated from the ascitic fluidusing ammonium sulphate at 45% (w/v) saturation. The precipitate wasdialysed against 50 mM Tris(pH 8.0) and then passaged through aProtein-A Sepharose column. The column was washed in 120 mM phosphatebuffer containing 0.15M NaCl until the optical density at 280 nm waszero. The proteins adsorbed by the Protein-A were eluted with 50 mMcitrate buffer (pH 6.0). The antibody was collected, dialysed againstidentical phosphate-buffered saline and stored at -40° C.. Usingconventional diffusion-in-agar-gel techniques, the antibody was shown tobelong to the IgG2a class. (For general discussion see Ref.11).

(ix) Collection and Purification of the Fib-75 Antigen from Human RedBlood Cells

Following the procedure described in Ref.12, the precipitated anddialysed antibody Fib-75-A was coupled to CNBr-activated Sepharose 4B at10 mg antibody protein per Gramme of beads. Red blood cells were washedand the membrane-ghosts prepared from them by lysis using 10 mM Tris (pH8.0). The ghosts were dissolved in 50 mM Tris (pH 8.0) containing 0.15 MNaCl and 1% sodium deoxycholate. The Sepharose-coupled FIB-75-A waspacked into a chromatography column, and the red blood cell ghost-lysatepumped through it. The column was washed until the optical density at280 nm was zero. Then the proteins adsorbed by the column were elutedwith 50 mM diethylamine buffer (pH 11.0). The protein peak was collectedand precipitated with acetic acid. The deoxycholate was removed usingethanol. This yielded approximately 200 μg of antigen per 10¹² red bloodcells.

Early studies indicate that the antigen was a glycoprotein with amolecular weight as determined by electrophoresis in polyacrylamide gelof approximately 19,500 daltons.

Later studies showed that the purified molecule had a molecular weightof 19,000±700D based on seven estimations on 10% SDS polyacrylamide gelsand appeared to be uncontaminated by other ghost proteins.

Amino-Acid Analysis

Samples (25 μg) of the purified antigen were hydrolysed in 6N HCl for 18hrs at 60° C. in evacuated glass tubes. The samples were freeze dried,dissolved in distilled water and frezze dried again prior to analysis ona Biotronic (Trade Mark) LC 2000 aminoacid analyser, using norleucine asan internal standard.

REACTIONS AND POTENTIAL OF FIB 75-A

The results from two independent preparations of the antigen are shownin Table 1. The results indicate that the protein is relatively rich inglutamate, aspartate, leucine and valine, relatively poor in methionineand contains no detectable proline.

Carbohydrate Analysis

Samples (100 μg) of the purified protein were analysed for their sugarcontent as described by Clamp et al (25) on a Perkin Elmer 200 gaschromatograph using D-mannitol as an internal standard.

The analysis (Table 2) from 3 independent preparations showed that theprotein was heavily glycosylated (approximately 30% by weight) and thatit was rich in galactose and glucosamine.

                  TABLE 1                                                         ______________________________________                                        Amino acid composition of the purified Fib 75.                                A binding glycoprotein.                                                                  Amino acids/100 residues                                           Amino acid   Preparation I                                                                            Preparation II                                        ______________________________________                                        Asx          18.6       16.4                                                  Glx          12.4       12.7                                                  Thr          6.7        6.4                                                   Ser          4.0        4.4                                                   Pro          0          0                                                     Gly          3.1        3.0                                                   Ala          5.7        5.9                                                   Cys          3.9        3.9                                                   Val          10.3       9.2                                                   Met          0.9        1.1                                                   Ileu         1.2        1.5                                                   Leu          10.0       10.0                                                  Tyr          3.73       4.4                                                   Phe          5.9        6.3                                                   Lys          8.0        8.4                                                   His          2.4        2.4                                                   Arg          3.1        3.6                                                   Trp          ND         ND                                                    ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        Sugar Composition of Purified Fib 75.                                         A Binding Protein                                                                     Carbohydrate nmols/mg protein                                         Sugar     Preparation I                                                                            Preparation II                                                                            Preparation III                              ______________________________________                                        Fucose    171        157         114                                          Mannose   269        266         169                                          Galactose 443        353         305                                          Glucose   36         30          57                                           Galactosamine                                                                           42         37          28                                           Glucosamine                                                                             621        515         463                                          Sialic acid*                                                                            108        98          95                                           % by weight                                                                             33.8       28.5        24.1                                         ______________________________________                                         *Possibly inaccurate                                                     

Immunohistochemical staining of a variety of human tissues by Fib-75-Ahas been conducted (Ref.5) and the results are summarised in Table 3.The epitope has been found to be present on most differentiated humannormal and neoplastic cells. All breast cancers examined reactedpositively although there was some heterogeneity of staining ofmalignant cells in tissue sections. However, there was uniform stainingof all malignant cells in effusions and tumour cell lines.

Of considerable importance is the absence of reaction with lymphocytesand colony forming units of human bone marrow.

                  TABLE 3                                                         ______________________________________                                        Human Tissues Reacting with Fib-75-A                                          ______________________________________                                        Positive   Neoplastic                                                                    Differentiated Human Tumour Cell                                              Lines* for example:                                                         MCF-7,MDA,                                                                    EJ, BARON       Refs. 13, 14, 17.                                             ZR, PAPIOU                                                                      Human Breast and other Carcinomas                                             Normal                                                                        Pulmonary Bronchioles and alveoli                                             Hepatic portal tracts                                                         Renal glomeruli and tubules                                                   Epidermis and appendages                                                      Blood vessels - endothelium                                                   Uterine smooth muscle                                                         Differentiated bone marrow elements:                                          Granulocytes, red blood cells.                                     Negative   Undifferentiated germ cell tumour                                             lines* for example:                                                          LICR-LONHT39/7                                                                                 Refs. 15, 16, 17.                                           Tera 1                                                                        Lymphocytes                                                                   Hepatic parenchyma                                                            Hypernephroma cell line (XK1)                                                 Colony Forming Units of Bone Marrow                                  ______________________________________                                         *All celllines cultured in vitro in DMEM buffered to 5% CO.sub.2 and          containing 10% foetal calf serum. No other additives required.           

IDENTIFICATION OF FIB-75 AND FIB-75-A

Binding to MFGM, as described in (vii) above, provides a convenientimmediate identification assay. Collection and identification of thecharacteristic antigen as a glycoprotein of 19,500 daltons shouldnormally be conclusive. A further cross check is provided by theimmunohistochemical reactions with human tissue summarized in Table 3,since this pattern of reactivity is common to no other known antibody.

Conclusive identification is possible via a competitive binding assay.As discussed in the introduction, the specific determinant or epitope,defining the precise region or structural configuration of the antigenmolecule identified by the antibody is not known and could reside in aspecific amino acid grouping or, for example, in a particularproteinaceous structural configuration. It is, therefore, in theorypossible for other closely similar antibodies to react with the sameantigen as obtained from red blood cells. Different antibodies would, ofcourse, be expected to recognize different epitopes and this differenceof epitopes can be recognized by conducting a competitive binding assayusing radioactively labelled bound antibody. Under equilibrium reactionconditions, the addition of unlabelled antibodies should displace aproportion of the labelled antibody which can then be determined. If thesuspect antibody is different, it will recognize a different epitopeand, therefore, the equilibrium conditions remain unchanged.

Naturally the 19,000 D glycoprotein antigen described above is peculiarto red blood cells. Antigens expressed by other cells e.g. breast orlung, could be identical or have different characteristics, especiallymolecular weight, since only the epitope or final determinant is common.

THERAPEUTIC POTENTIAL OF FIB-75

During the past decade, there has been an increased appreciation of theimportance of accurate staging of patients with cancer at the time oftheir initial presentation. This emphasis has resulted in the design oftherapeutic regimes more appropriate to the stage of disease than waspossible hitherto.

The Applicants have had an interest, for some considerable period oftime, in trying to detect "latent" metastases in patients with operablebreast cancer at the time of their initial presentation. For thispurpose a variety of biochemical methods were used to measure tumourmarkers predominantly in the plasma but also in the urine. The resultsof those studies showed conclusively that many patients with overtmetastases (end-stage disease) had abnormal tumour marker levels.However, few patients considered to have operable localised primarytumours, but whose subsequent course showed that micrometastases musthave been present at that time, had raised values. In longitudinalstudies, it was also found that while elevated values did occur in manyinstances before the overt manifestation of metastases, the lead timecreated was of the order of only 4-5 months, a time that has no clinicalutility.

Accordingly, attempts have been made by the Applicants and theirassociates (Ref.24) to evolve alternative methods to detect metastaticdisease. In this context, morphological immunocytochemical methods havebeen used to detect tumour cells in the bone marrow of breast cancerpatients at the time of their initial presentation. These results havebeen fully documented in Ref.17 (p.24). Using antibodies to theepithelial membrane antigen (EMA) or more recently monoclonal antibodiesto breast cell surfaces (LICR-LON-M8), between 20-30% of patientswithout apparent evidence of spread beyond the axillary lymph nodes atthe time of their initial presentation have been found to have tumourcells in their bone marrow. Preliminary follow-up of those patients hasshown overt metastases earlier than in subjects in whom such cells arenot demonstrable.

It is hoped that this will provide a new approach to treating breastcancer patients with `small volume` disease. One regimen would be tosubject the patients to a form of high dose chemotherapy. This wouldkill not only the tumour cells but also the normal marrow population.Hence this therapy only becomes reasonable and rational if it ispossible to remove tumour-containing bone marrow from those patients,kill the tumour cells in vitro and then return the cleaned-up bonemarrow to the patients once the high dose therapy has been completed.

The therapeutic value of many cytotoxic drugs is limited by theirtoxicity to heamatopoietic stem cells in the bone marrow. Much largerdoses can be given safely if the marrow is harvested beforeadministration of the cytotoxic agent and then reinfused once the drughas been excreted or metabolised. This technique, autologous marrowrescue, has been used to allow administration of high doses ofmelphalan, cyclophosphamide, BCNU and VP-16-213 in many tumoursincluding leukaemias, melanoma, neuroblastoma and several other solidtumours. However, even if such high-dose therapy is effective, viabletumour cells may be reintroduced into the patient on reinfusion if themarrow is infiltrated by malignant cells which may then provide a sourcefor future relapse.

In those tumours where bone marrow infiltration is common, there is,therefore, considerable interest in a `clean up` procedure to eliminatemalignant cells whilst sparing the bone marrow stem-cells. In theleukemias, such cleaning-up procedures have been carried out usingconventional (Ref.18) and monoclonal antibodies (Ref.19) (both withheterologous complement) and with certain cytotoxic drugs (Ref.20).

In this context the monoclonal antibody Fib-75-A may have an importantclinical role, since it does not react with colony forming units ofnormal human bone marrow but has specific cytotoxicity to a number ofhuman epithelial tumour cell lines. Accordingly, cytotoxicity studieswere conducted in the absence and presence of bone marrow cells.

Complement Mediated Cytotoxicity

51Cr-release assays according to the method of Brunner et al (Ref.21)together with Trypan Blue exclusive (i.e. viability) estimates wereemployed in the presence of rabbit complement (RC) (Pel-Freez) to accessthe degree of cytotoxicity. After preliminary experiments with 1×10⁵MCF-7 (breast), MDA (Breast) or EJ (Bladder) cells as targets in thepresence of 10⁷ bone marrow cells, maximal cell killing was found to beachieved by incubation at 37° C. for 2 hours in the presence of 5 μgFib-75-A, and aliquots of 200 μl RC added at the beginning and after 1hour.

Using the above quantities, cytotoxicity was examined on cell lines andmalignant effusions (Table 4). The results indicate that no tumour cellsare viable after exposure to Fib-75-A and complement except when largeclumps (>500 cells) are present.

                                      TABLE 4                                     __________________________________________________________________________    CYTOTOXIC EFFECT OF FIB-75 PLUS RABBIT COMPLEMENT ON CELL                     LINES AND MALIGNANT BREAST CANCER CELLS IN EFFUSIONS.sup.(a)                                                            % .sup.51 Cr Release                                                          Tumour Cells +                                                                          Tumour Cell               Cell             Number of         Tumour Cells                                                                         Bone Marrow                                                                             Viability by              Source           Experiments       Alone  Cells.sup.(b)                                                                           Tryptan                   __________________________________________________________________________                                                        Blue                      MALIGNANT CELL                                                                LINES                                                                         MCF-7            5                 84     ND        0%                                                           (76-92)                                    MDA              16                83     77        0%                                                            (68-100)                                                                            (59-84)                             EJ               5                 73     65        0%                                                           (69-76)                                                                              (62-68)                             MALIGNANT EFFUSIONS     % MALIGNANT                                           (BREAST CANCER)         CELLS IN FLUID                                        Patient A        2      >90%       80     ND        0%                        Patient B                                                                     1st Ascites      1       40%       25     ND        55.sup.(e)                2nd Ascites      1      >80%       64     52        0%                        Patient C        1       90%       55     UNINT.sup.(c)                                                                           5%                        Patient D        1      ?          66     51        LARGE                     __________________________________________________________________________                                                        CLUMPS.sup.(d)             Footnotes to Table 4:                                                         .sup.(a) The amount of .sup.51 Crreleased into the supernatant (in counts     per minute (cmp)) was expressed as a percentage of the maximum release        achieved by lysis with 5% NP40 detergent according to the formula:            ##STR1##                                                                      As a control for this experiment cells were incubated with antibody alone     and with complement alone. These assays were done in triplicate. In all       these experiments 1-5 × 10.sup.5 target cells were used in a            standard volume of 500 μl of tissue culture medium (RPMI 1640 with 2%      foetal calf serum).                                                           .sup.(b) Nucleated bone marrow cells from human donors were collected in      early experiments by separating whole bone marrow over a FicollHypaque        mixture of density 1 · 100 g/ml. This was found to retain most       nucleated cells including granulocytes at the interface. 5-10 ×         10.sup.5 nucleated bone marrow cells were resuspended in 0.5 ml medium an     5-10 × 10.sup.4 labelled target cells added to represent bone marro     infiltrated with 1% of malignant cells. Subsequent results in CFUC assays     showed better preservation if the granulocytes were eliminated by using       FicollHypaque of density 1 · 077 (`Lymphoprep`).                     .sup.(c) Uninterpretable due to very high spontaneous loss of .sup.51 Cr.     .sup.(d) Some large clumps (>500 cells) contained some viable cells.          .sup.(e) Contamination by mesothelial cells.                             

Effect on Bone Marrow `Stem` Cells

With the demonstration of tumour cell killing, it was important toascertain if the therapy spared the bone marrow cells thereby enablingpotential in vivo recovery.

Bone marrow stem-cells were assayed by the standard colony-forming-unit(CFU-C) method (Ref.22) which measures progenitor cells committed to thegranulocyte-macrophage series, and also by pluripotential stem-cellassays on CFU-C, erythroid stem-cells (BFU-E), megakaryocytic (Megakar)colony-forming units and mixed colonies of erythroid-myeloid types(Ref.23). The results are shown in Table 5.

                  TABLE 5                                                         ______________________________________                                        EFFECT OF FIB-75 AND COMPLEMENT ON                                            PLURIPOTENTIAL STEM-CELL ASSAY                                                Aliquots of                                                                              Bone marrow                                                        complement % survival of                                                      added      CFU-C    BFU-E     MegaKar                                                                              Mixed                                    ______________________________________                                        1          36       48        22     72                                       2          52       45        33     10                                       1          95       52        87     25                                       2          85       47        62     100                                      1          102      70        75     75                                       2          90       81        100    200                                      ______________________________________                                    

It may be concluded from Table 3 that while CFU-C's may be reduced toapproximately 60%, the effect of Fib-75-A and RC as assessed in thepluripotential assay was insignificant.

Effect on Malignant Colony-Forming Cells

Once the ⁵¹ Cr-release assays had shown maximal cell killing, platingassays were done to identify any remaining tumour cells still capable ofdivision. EJ (bladder) cells were used because of their high platingefficiency. A minimum of two cells are required to form one identifiablecolony at one week. EJ cells were treated with Fib-75-A and complementin suspension, then plated into tissue cultures wells in full growthmedium to detect any residual cells capable of division and colonyformation.

With aliquots of 1×10⁵ cells with and without the presence of bonemarrow cells, no colonies were seen after 7 days at which time allcontrol wells had achieved confluent growth.

SUMMARY

Autologous marrow rescue is an essential component of high-dose therapy,using either radiation of chemotherapy but it has been recognized thatmany patients at initial presentation with solid tumours, includingbreast (Ref.24) and lung carcinomas have marrow infiltration. Sincereinfusion of these tumour cells could provide foci for recurrentdisease, ways of killing these cells have been investigated.

Complement lysis of tumour cells in vitro has been demonstrated usingthe Fib-75 antibody. In order to ascertain whether all cancer cells arekilled, the experiments included the highly clonogenic cell line, EJ,derived from a human bladder carcinoma and it was demonstrated thatclonogenicity was abolished up to a contamination of 10⁵ EJ cells in 10⁷bone marrow cells. Using the same quantities of antibody and complementon malignant cells derived from patients' effusions, which cannot begrown readily in culture, more than 95% of the cancer cells were killedalthough large clumps of tumour cells were found to contain viable cellsafter exposure, presumably due to inadequate penetration of antibody.This problem relevant to the treatment of infiltrated bone marrow, sincesuch large clumps of tumour cells do not occur at this site.

The antibody also binds human complement and although this was arelatively unreliable source of complement or routine assays it doessuggest that if any tumour cells binding Fib-75-A are viable at the timeof re-infusion, they are likely to be killed in the presence of freshcomplement in the patient's plasma.

The quantities of Fib-75-A and rabbit complement used to kill malignantcells depressed the bone marrow CFU-Cs by less than 50%. However theCFU-C assay measures a stem-cell committed to the granulocyte-macrophageseries and which is thought to be relatively advanced in thedifferentiation process. Since mature granulocytes express the Fib-75antigen strongly it is likely that effect on the CFU-C assay is greaterthan the effect on the true totipotential stem-cell which cannot, atpresent, be assayed. The pluripotential stem-cell assays performed tendto support that idea that the less differentiated earlier stem-cells arerelatively unaffected.

Absorption of rabbit complement against human blood cells was shown tobe necessary in order to prevent the occasional very severe effect onthe CFU-C assay which was probably due to heterophile antibody in therabbit serum and which would therefore be expected to have anon-specific toxic effect on all bone marrow stem-cells.

The results indicated that Fib-75-A mediates complement lysis of tumourcells in bone marrow both effectively and safely. The antibody is notspecific to breast carcinoma and could therefore be used in all tumoursthat bear the Fib-75 antigen provided that tumour cells in the marrow donot occur in very large clumps. The results are sufficiently encouragingto permit the commencement of a high dose therapy programme for breastcancer patients.

Further therapeutic potential

Studies are in hand to conjugate a toxin (e.g. ricin) to Fib-75-A. Theantibody-toxin conjugate will be assessed for its effect on model breasttumour cell lines in vitro and its effect on the colony-forming-unitcapacity. It is hoped that this will obviate the use of large amounts ofrabbit complement.

It is further envisaged that Fib-75-A, in association with complement,or as an antibody-toxin conjugate, may provide a valuable reagent foraiding autologous bone marrow grafting in patients, in particular withbreast and oat cell carcinoma, at the time of their initial presentationwhen they are found to have micrometastatic disease.

Where national jurisdiction permits, the invention is inclusive ofmethods of therapeutic and diagnostic treatment of humans or animalsutilizing the antibody Fib-75-A as described above. In particular theinvention is inclusive of a method of treating cancer (especially cancerof the breast) which comprises harvesting a sample of bone marrow from apatient, subjecting the patient to treatment adapted to kill allcancerous material at least within the bone marrow, subjecting some orall of the samples to cytotoxic treatment with Fib-75-A (or a toxinconjugate thereof) adapted to kill cancerous cell lines while leavingviable colony-forming-units of bone marrow, and reintroducing thetreated sample material to the blood stream of the patient.

Toxins suitable for conjugation may include e.g. macromolecular toxinssuch as ricin, abrin, or subunits thereof, drugs such as for examplemethotrexate and adriamycin, and radio-active isotopes such as iodine131 and indium 111. Bound isotopes are also useful for localisation.Conjugation of antibodies of a similar nature with toxins is a knownprocedure cf. Ross et al (26). Conjugation with isotopes is straightforward by well established chemical reactions.

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We claim:
 1. A biologically pure culture of the monoclonal hybridomadesignated LICR-LON Fib 75 (CNCM, I-22).
 2. The antibody designatedLICR-LON-Fib 75 as generated by the hybridoma culture of claim 1.